Human parainfluenza type 2 virus (hPIV2) infects human respiratory mucosa and can induce mucosal immunity, and therefore is expected to find application as a vaccine vector. In order to put this vector to practical use, the virus vector is required to have an ability to infect human cells and not to generate transmissible viruses in the human body after infection (such a vector is referred to as a non-proliferative vector). Thus a system is required which can primarily infect cells or tissue but does not produce transmissible viruses in the infected cells or tissue so that it does not cause recurrent infections. The procedure for construction of such a system generally includes partially deleting a gene on a viral genome, preparing cells expressing a product encoded by the deleted gene, and complementing in trans the defective virus in the cells with the product encoded by the deleted gene to prepare a non-proliferative vector. This system has been introduced to DNA viruses such as adenoviruses (e.g. WO 94/28152) and RNA viruses such as retroviruses (e.g. WO 2006/084746). For viruses of Paramyxoviridae to which human parainfluenza type 2 virus belongs, a Sendai virus vector defective in the F gene and the like has been proposed (WO 2000/070070). It has also been reported that a vaccine for the prevention of onset of tuberculosis, which utilizes a non-proliferative recombinant vector derived from human parainfluenza type 2 virus by incorporating an a antigen (Ag85B) gene from typical mycobacteria such as Mycobacterium kansasii or Mycobacterium bovis BCG (PCT/JP 2010/069435), has a high suppressing effect of Mycobacterium tuberculosis proliferation.
F gene-deficient human parainfluenza type 2 virus does not contain, in itself, the Fusion protein (hereinafter referred to as “F protein”) of human parainfluenza type 2 virus which is required for, during viral replication and transcription after invasion into a host cell, fusing a viral envelope with a cell membrane and introducing a viral nucleocapsid into the host cell, and thus cannot form replication competent viral particles. Therefore the preparation of a non-proliferative human parainfluenza type 2 virus vector which is defective in the F gene on the genome but contains the F protein on a vector envelope includes generating cells expressing the F protein of the virus, culturing the defective virus on the cells in order to retain the F protein on the viral envelope in the presence of the F protein complemented in trans by the cells and thereby constructing infectious viral particles. The human parainfluenza type 2 virus particles collected from the culture supernatant prepared according to the above replication system contain a genome lacking the F gene. Incorporating therapeutic genes or genes encoding vaccine antigens into the F gene-defective human parainfluenza type 2 virus vector may provide viral particles useful as medicaments.
Because viruses generally include membrane proteins which are cytotoxic, it has been conventionally necessary to suppress expression of the viral F gene and the like at normal times by establishing a cell line after introducing a vector which is designed to express the F gene and the like under the control of an inducible promoter and to induce the expression of the F gene and the like only when the virus is reconstituted after infecting helper cells which express the F gene and the like. In the above document pertaining to the Sendai virus vector (WO 2000/070070), for example, a system is used in which the F gene of Sendai virus is not expressed during proliferation of host cells by using the Cre-loxP inducible system and the expression thereof is induced upon infection of the virus to the cells by addition of an adenovirus.
However, the step of preparation and addition of an adenoviruses makes commercial production of the virus vector complicated. There is also a problem in that the medicament manufacturing control step becomes complicated because the gene introducing efficacy by an adenovirus is not constant. Further there is a problem in that during actual production of the virus vector with the membrane protein expression inducible system by culturing cells and replicating viral particles, the viral replication capacity decreases with an increase in the number of passages of the host cells due to toxicity of the expressed membrane protein, such that the cells lose the production ability of the virus after about 5 passages.
Therefore there is a need to obtain, as a cell which can express a gene encoding a membrane protein and the like that is defective in a virus and allows replication of the defective virus vector, a cell system which can constitutively and stably express the protein. There is also a need for a host cell system which has such robustness that the properties thereof are stable after subculturing.
The references cited in the present specification are shown below. The contents of these references are incorporated herein by reference in their entirety. However it does not intend to admit that any of these references is available as “Prior Art” to the present specification.
Patent Document 1: WO 94/28152
Patent Document 2: WO 2006/084746
Patent Document 3: WO 2000/070070
Patent Document 4: PCT/JP 2010/069435